The invention relates to Vitamin D3 analogs, particularly 20epi-16-ene analogs of Vitamin D3.
The invention relates to compounds of the formula 
wherein R is hydrogen, fluorine, or hydroxyl, R2 is lower alkyl or C(R3)3 and R3 is halogen, X is xe2x95x90CH2 or when R is hydroxy, X is hydrogen or xe2x95x90CH2,
and A is xe2x80x94Cxe2x89xa1Cxe2x80x94, 
or
xe2x80x94CH2xe2x80x94CH2xe2x80x94, provided that when A is xe2x80x94CH2xe2x80x94CH2xe2x80x94, R2 is lower alkyl.
Compounds of formula I induce differentiation and inhibition of proliferation in various skin and cancer cell lines. Accordingly, the compounds of formula I are useful as agents for the treatment of hyperproliferative skin diseases, such as, psoriasis. Compounds of formula I are also useful in the treatment of neoplastic diseases, such as, leukemia or breast cancer and sebaceous gland diseases, such as, acne or seborrheic dermatitis.
As used herein, xe2x80x9clower alkylxe2x80x9d denotes a straight or branched chain alkyl group containing 1 to 4 carbon atoms, for example, methyl, ethyl, propyl, isopropyl, butyl, t-butyl and the like. Preferably lower alkyl is methyl or ethyl. Halogen means fluorine, iodine, bromine or chlorine, preferably, fluorine.
In the formulas presented herein, the various substituents are illustrated as joined to the nucleus by one of the following notations: a wedged solid line () indicating a substituent which is above the plane of the molecule, (xcex2-orientation) and a wedged dotted line () indicating a substituent which is below the plane of the molecule (xcex1-orientation).
As used herein, the term xe2x80x9cExe2x80x9d denotes 
that is, a stereochemical configuration about a carbon-carbon double bond, such that the two hydrogens are attached to different carbon atoms, and are on opposite sides of the carbon-carbon double bond.
The term xe2x80x9cZxe2x80x9d denotes 
that is a stereochemical configuration about a carbon-carbon double bond, such that the two hydrogens are attached to different carbon atoms and are on the same side of the carbon-carbon double bond.
The invention relates to compounds of the formula 
wherein R is hydrogen, fluorine, or hydroxyl, R2 is lower alkyl or C(R3)3 and R3 is halogen, X is xe2x95x90CH2, or when R is hydroxy X is hydrogen or xe2x95x90CH2, and A is xe2x80x94cxe2x89xa1cxe2x80x94, 
or
xe2x80x94CH2xe2x80x94CH2xe2x80x94, provided that when A is xe2x80x94CH2xe2x80x94CH2xe2x80x94, R2 is lower alkyl.
Compounds of formula I induce differentiation and inhibition of proliferation in various skin and cancer cell lines. Accordingly, the compounds of formula I are useful as agents for the treatment of hyperproliferative skin diseases, such as, psoriasis. Compounds of formula I are also useful in the treatment of neoplastic diseases, such as, leukemia or breast cancer and sebaceous gland diseases, such as, acne or seborrheic dermatitis. Compounds of formula I, particularly 1xcex1-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-chole-calciferol, are useful in the treatment of osteoporosis, see U.S. copending patent application Ser. No. 08/857,569, filed May 16, 1997, which is incorporated by reference herein.
The invention also relates to a pharmaceutical composition comprising a compound of formula I and a method of treating neoplastic diseases, sebaceous gland diseases and hyperproliferative skin diseases by administration of a compound of formula I.
The invention also relates to a process for preparing compounds of formula I and intermediates of formula XII.
In a preferred embodiment, R is hydroxyl. In a compound of formula I, R2 is preferably hydrogen or fluorine. In a preferred compound of formula I, A is double bond or triple bond or single bond. Preferred compounds of formula I are:
1,25-dihydroxy-16-ene-23-yne-20-epi-cholecalciferol;
1,25-dihydroxy-16-ene-20-epi-cholecalciferol;
1,25-dihydroxy-16-ene-23-yne-26,27-hexafluoro-20-epi-cholecalciferol;
1xcex1-fluoro-25-hydroxy-16,23Z-diene-26,27-hexafluoro20-epi-cholecalciferol;
1xcex1-fluoro-25-hydroxy-16-ene-23-yne-26,27-hexafluoro20-epi-cholecalciferol;
1,25-dihydroxy-16,23E-diene-26,27-hexafluoro-20-epi-cholecalciferol;
1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-20-epi-cholecalciferol;
1,25-dihydroxy-16,23Z-diene-19-nor-26,27-hexafluoro-20epi-cholecalciferol.
The compounds of formula I are prepared as hereafter described in Schemes I-V and the Examples. 
wherein R2 is as described above.
In above Scheme I, the compound of formula II, a known compound (B. M. Trost, P. R. Bernstein, P. C. Funfschilling, J. American Chemical Society 101, 4378 (1979)), is converted to the compound of formula III by reaction with acetic anhydride, pyridine and dimethylaminopyridine in a chlorinated hydrocarbon solvent, such as dichloromethane. The reaction is carried out at 0xc2x0 C. to 50xc2x0 C., preferably room temperature, preferably under an argon atmosphere.
The compound of formula III is converted to the compound of formula IV by reaction with a base, such as, sodium carbonate in an alcohol solvent, such as methanol, under, preferably, an argon atmosphere.
The compound of formula IV is converted to the compound of formula V by reaction with oxalyl chloride and dimethylsulfoxide in a chlorinated hydrocarbon solvent, such as, dichloromethane, under an argon atmosphere.
The compound of formula V is converted to the compound of formula VI by reaction with benzalacetone in the presence of palladium on charcoal catalyst.
The compound of formula VI is converted to the compound of formula VII by reaction with a 3-trimethylsilylpropynal and a Lewis acid such as dimethylaluminum chloride in a chlorinated hydrocarbon solvent, such as dichloromethane.
The compound of formula VII is converted to the compound of formula VIII by reaction of the corresponding phenylthiono-carbonate with tri-n-butylhydride and triethylborane in toluene.
The compound of formula VIII is converted to the compound of formula IX by reaction with a base, such as, sodium hydroxide in an alcohol solvent, such as ethanol. The reaction is carried out at a temperature in the range of 50xc2x0 C. to 100xc2x0 C., preferably 80xc2x0 C.
The compound of formula IX is converted to the compound of formula X by reaction with 1-(trimethylsilyl)imidazole in a chlorinated hydrocarbon solvent, such as anhydrous methylene chloride.
The compound of formula X is converted to a compound of formula XI by reaction with the corresponding compound
of formula 
wherein R2xe2x80x2 is hydrogen, or lower alkyl in the presence of a base such as n-butyllithium.
The reaction is preferably carried out at xe2x88x9278xc2x0 C.
When the compounds of formula I wherein R2 is halogen are prepared, the compound of formula X is reacted with a halogenated acetone, such as hexafluoroacetone in the presence of a base such as n-butyllithium.
The compound of formula XI is converted to the corresponding compound of formula XII by reaction with tetrabutylammonium fluoride in an ether solvent such as, tetrahydrofuran. 
wherein R, X and R2 are as described above.
As set forth in Scheme II, a compound of formula XII is converted to a corresponding compound of formula XIII by reaction with an oxidizing agent such as, pyridinium dichromate in a chlorinated hydrocarbon solvent such as, anhydrous methylene chloride.
A compound of formula XIII is converted to a corresponding compound of formula XIV by reaction with 1-(trimethylsilyl)imidazole in a chlorinated hydrocarbon solvent, such as, anhydrous methylene chloride.
A compound of formula XIV is converted to a corresponding compound of formula Ia wherein R is hydroxy and X is xe2x95x90CH2, by reaction with [3S-(1Z,3xcex1,5xcex2)]-[2-[3,5-bis[[1,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylene-cyclohexylidene]ethyl]diphenyl-phosphine oxide in an ether like solvent such as tetrahydrofuran in the presence of n-butyllithium as a base.
Alternatively, a compound of formula XIV is converted to the corresponding compound of formula Ia wherein R is hydrogen by reaction with [5S,Z]-2-[2-[2-methylene-5-[[(1,1-dimethylethyl)-dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in an ether like solvent such as tetrahydrofuran in the presence of n-butyllithium as a base.
Alternatively, a compound of formula XIV is converted to the corresponding compound of formula Ia wherein R is fluorine by reaction with [3S-(3xcex1,5xcex2,Z)]-2-[2-[2-methylene-3-fluoro-5-[[( 1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenyl-phosphine oxide in a ether like solvent such as tetrahydrofuran in the presence of n-butyllithium as a base.
Alternatively, a compound of formula XIV is converted to the corresponding compound of formula Ia wherein R is hydroxy and X is hydrogen by reaction with [3R-(3xcex1,5xcex2,Z)-3,5-bis[[(1,1-dimethylethyl)-dimethylsilyl]oxyl]cyclohexylidene]ethyl]diphenylphosphine oxide in an ether like solvent, such as tetrahydrofuran in the presence of nbutyllithium as a base. 
wherein R, X are as described above and R2xe2x80x2 is hydrogen or lower alkyl.
In the above Scheme III, a compound of formula XII is reduced to a corresponding compound of formula XV by reaction with hydrogen and Lindlar catalyst in an organic solvent, such as, a combination of ethyl acetate, hexane and ethanol, in the presence of quinoline.
A compound of formula XV is converted to a corresponding compound of formula XVI by reaction with hydrogen in the presence of a catalyst such as 1,4-bis(diphenylphosphino)butane 1,5-cyclooctadiene rhodium tetrafluoroborate and mercury in a chlorinated hydrocarbon solvent, such as, methylene chloride.
A compound of formula XVI is oxidized to a corresponding compound of formula XVII by reaction with pyridinium dichromate in a chlorinated hydrocarbon solvent, such as methylene chloride.
A compound of formula XVII is converted to a corresponding compound of formula XVIII by reaction with 1-(trimethylsilyl)-imidazole in a chlorinated hydrocarbon solvent, such as methylene chloride.
A compound of formula XVIII is converted to a corresponding compound of formula Ib wherein R is hydroxy and X is xe2x95x90CH2, by reaction with [3S-(1Z,3xcex1,5xcex2)]-[2-[3,5-bis[[1,1-dimethylethyl)-dimethylsilyl]oxy)-2-methylene-cyclohexylidene]ethyl]diphenyl phosphine oxide in the presence of n-butyllithium as a base, preferably at a temperature of xe2x88x9278xc2x0 C. in anhydrous tetrahydrofuran as solvent.
Alternatively, a compound of formula XVIII is converted to a corresponding compound of formula Ib wherein R is hydroxy and X is hydrogen by reaction with [3R-(3xcex1,5xcex2,Z)-3,5-bis[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in the presence of n-butyllithium as a base in anhydrous tetrahydrofuran as a solvent.
Alternatively, a compound of formula XVIII is converted to the corresponding compound of formula Ib wherein R is fluorine by reaction with [3S-(3xcex1,5xcex2,Z)]-2-[2-[2-[methylene-3-fluoro-5-[[1,1 dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in the presence of n-butyllithium as a base in anhydrous tetrahydrofuran as a solvent, at a temperature of xe2x88x9278xc2x0 C.
Alternatively, a compound of formula XVIII is converted to the corresponding compound of formula Ib wherein R is hydrogen by reaction with [5S,Z]-2-[2-[2-methylene-5-[[1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in the presence of n-butyllithium as a base in anhydrous tetrahydro-furan as a solvent at a temperature of xe2x88x9278xc2x0 C. 
wherein R, X and R2 are as described above.
In above Scheme IV, a compound of formula XII is converted to a corresponding compound of formula XIX by reaction with a reducing agent such as, lithium aluminum hydride in an ether solvent, such as, tetrahydrofuran in the presence of sodium methoxide as a base.
A compound of formula XIX is converted to a corresponding compound of formula XX by reaction with an oxidizing agent such as,pyridinium dichromate in a chlorinated hydrocarbon solvent, such as, methylene chloride.
A compound of formula XX is converted to a corresponding compound of formula XXI by reaction with 1-(trimethylsilyl)-imidazole in a chlorinated hydrocarbon solvent, such as, methylene chloride.
A compound of formula XXI is converted to a corresponding compound of formula Ic wherein R is hydroxy and X is xe2x95x90CH2, by reaction with [3S-(1Z,3xcex1,5xcex2)]-[2-[3,5-bis[[1,1-dimethylethyl)-dimethylsilyl]oxy]-2-methylene-cyclohexylidene]ethyl]diphenyl-phosphine oxide in an ether solvent, such as, tetrahydrofuran, in the presence of a base, such as n-butyllithium.
Alternatively, a compound of formula XXI is converted to a corresponding compound of formula Ic wherein R is hydroxy and X is hydrogen by reaction with [3R-(3xcex1,5xcex2,Z)]-3,5-bis[[1,1-dimethyl-ethyl)-dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in an ether solvent, such as tetrahydrofuran, in the presence of a base, such as n-butyllithium.
Alternatively, a compound of formula XXI is converted to a corresponding compound of formula Ic wherein R is hydrogen by reaction with [5S,Z]-2-[2-[2-methylene-5-[[1,1-dimethylethyl)-dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in an ether like solvent such as, tetrahydrofuran, in the presence of a base such as n-butyllithium.
Alternatively, a compound of formula XXI is converted to a corresponding compound of formula Ic wherein R is fluorine by reaction with [3S-(3xcex1,5xcex2,Z)]-2-[2-[2-methylene-3-fluoro-5-[[(1,1dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in a ether like solvent, such as tetrahydrofuran, in the presence of a base such as n-butyllithium. 
wherein R, X and R2 are as described above.
As set forth in Scheme V above, a compound of formula XII is converted to a corresponding compound of formula XXII by hydrogenation with Lindlar catalyst in the presence of quinoline in a mixture of solvents, such as a combination of ethyl acetate, hexane and ethanol.
A compound of formula XXII is converted to a corresponding compound of formula XXIII by reaction with oxidizing agent, such as, pyridinium dichromate in a chlorinated hydrocarbon solvent, such as, methylene chloride.
A compound of formula XXIII is converted to a corresponding compound of formula XXIV by reaction with 1-(trimethylsilyl)imidazole in a chlorinated hydrocarbon solvent, such as, methylene chloride.
A compound of formula XXIV is converted to the corresponding compound of formula Id wherein R is hydroxy and X is CH2, by reaction with [3S-(1Z ,3xcex1,5xcex2)]-[2-[3,5-bis [[1,1-dimethylethyl)dimethylsilyl]oxy]-2-methylenecyclohexylidene]ethyl]diphenylphosphine oxide in an ether solvent, such as tetrahydrofuran, in the presence of a base such as butyl lithium.
Alternatively, a compound of formula XXIV is converted to the corresponding compound of formula Id wherein R is hydroxy and X is hydrogen by reaction with [3R-(3xcex1,5xcex2,Z)]-3,5-bis [[(1,1-dimethyl-ethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in tetrahydrofuran in the presence of a base such as n-butyllithium.
Alternatively, a compound of formula XXIV is converted to the corresponding compound of formula Id wherein R is hydrogen by reaction with [5S,Z]-2-[2-[2-methylene-5-[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in an ether like solvent such as tetrahydrofuran in the presence of a base such as n-butyllithium.
Alternatively, a compound of formula XXIV is converted to the corresponding compound of formula Id wherein R is fluorine by reaction with [3S-(3xcex1,5xcex2,Z)]-2-[2-[2-methylene-3-fluoro-5-[[(1,1-dimethylethyl)dimethylsilyl]oxy]cyclohexylidene]ethyl]diphenylphosphine oxide in an ether like solvent such as tetrahydrofuran in the presence of a base such as n-butyllithium.
The compounds of formula I can be administered orally, for example, in the form of tablets, coated tablets, dragees, hard or soft gelatin capsules, solutions, emulsions or suspensions. The administration can, however, also be effected rectally, for example, in the form of suppositories or parenterally, for example, in the form of injection solutions.
A composition in accordance with the invention can be processed with pharmaceutically inert, inorganic or organic excipients for the manufacture of tablets, coated tablets, dragees and hard gelatin capsules. Lactose, corn starch or derivatives thereof, talc, stearic acid or its salts, and the like, can be used as such excipients, for example, for tablets, dragees and hard gelatin capsules.
Suitable excipients for soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solid and liquid polyols, and the like; depending on the nature of the active ingredient. No excipients are, however, usually required in the case of soft gelatin capsules.
Suitable excipients for the preparation of solutions and syrups, are, for example, water, polyols, saccharose, invert sugar, glucose and the like.
Suitable excipients for injection solutions are, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
Suitable excipients for suppositories are, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols and the like.
Moreover, the pharmaceutical preparations can contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorants, salts for varying the osmotic pressure, buffers, masking agents or antioxidants.
The compounds of formula I as described above can be administered orally or by injection, for the treatment of neoplastic diseases such as leukemia, to warmblooded animals which need such treatment. More specifically, the compounds of formula I as described above can be administered orally to an adult human in dosages that are in the range of about 0.25 to 50 xcexcg per day for the treatment of neoplastic diseases such as leukemia or breast cancer.
The compounds of formula I as described above can be administered orally, for the treatment of hyperproliferative skin diseases such as psoriasis, basal cell carcinomas, disorders of keratinization, and keratosis, to warmblooded animals which need such treatment. More specifically, the compounds of formula I as described above can be administered orally to an adult human in dosages that are in the range of about 0.25 to 50 xcexcg per day for the treatment of hyperproliferative skin diseases such as psoriasis, basal cell carcinomas, disorders of keratinization, and keratosis. These compounds can be administered orally for the treatment of acne in humans at a dosage of about 0.25 to 50 xcexcg per day; preferably 0.5 to 5 xcexcg per day.
The compounds of formula I as described above can be administered topically, for the treatment of hyperproliferative skin diseases such as psoriasis, basal cell carcinomas, disorders of keratinization, and keratosis, to warmblooded animals which need such treatment. More specifically, the compounds of formula I as described above can be administered topically in dosages that are in the range of about 0.5 to about 100 xcexcg per gram of topical formulation per day, for the treatment of hyperproliferative skin diseases such as psoriasis, basal cell carcinomas, disorders of keratinization, and keratosis.
The compounds of formula I as described above can also be administered topically for the treatment of sebaceous gland diseases such as acne or seborrheic dermatitis.
The useful activity of compounds of formula I as agents for the treatment of neoplastic diseases can be demonstrated by the following test procedures.
HL-60 Cell Differentiation
The induction of differentiation of HL-60 cells was assayed by measuring their oxidative burst potential via the reduction of nitrobluetetrazolium (NBT).
HL-60 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 1 mM sodium pyruvate, 1% non-essential amino acids, 50 U/ml penicillin and 50 xcexcg/ml streptomycin (RPMI/FCS). HL-60 cells (30,000 cells/90 xcexcl of RPMI/supplemented medium) were seeded into flat-bottomed microtiter wells. Immediately after seeding, 10 xcexcl of test compounds diluted in supplemented RPMI medium were added to the wells at the same time to yield final concentrations between 10xe2x88x9211 and 10xe2x88x926 M (starting from stock solutions of 10xe2x88x922 M in ethanol stored at xe2x88x9220xc2x0 C. and protected from light). After 3 days, the medium was removed from the wells with a multichannel pipette and replaced with 100 xcexcl of NBT solution (1 mg/ml in phosphate buffered saline (PBS) with 200 nM phorbol myristate acetate (PMA). Following an additional hour incubation at 37xc2x0 C. the NBT solution was removed and 100 xcexcl of 10% sodium dodecyl sulfate (SDS) in 0.01 N HCl was added. The amount of the reduced NBT was quantified photometrically at 540 nm using an automated plate reader. The mean of 3 wells was calculated. S.E.M. were between 5 and 10%. Values were expressed as percent of maximal differentiation achieved with 100-1000 nM calcitriol in the same experiment. The concentration (nM) leading to 50% of this maximal value is determined graphically and given in Table I as ED50.
The two cell lines used in these experiments and their growth requirements are listed below:
1. T47-D breast carcinoma cells were grown in RPMI 1640 medium supplemented with 10 xcexcg/ml bovine insulin, and 10% fetal bovine.
2. MCF-7 breast carcinoma cells were grown in MEM (Eagles) supplemented with non-essential amino acids, 1 mM sodium pyruvate, 10 xcexcg/ml bovine insulin, and 10% fetal bovine serum.
Cells were grown in appropriate medium to late log phase (xcx9c80% confluency). T47-D or MCF-7 cells are then trypsinized and seeded at 4000 or 2000 cells/well, respectively. At 24 hour post seeding, serial dilution of ethanol-solubilized drugs are prepared in the same medium and added to triplicate wells at a final concentration of 1,000 to 0.1 nM and 0.1% ethanol. On day 3 to 7 post drug addition, 50 xcexcl of a 5 mg/ml MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide in phosphate-buffered saline) is added to each well and incubation is continued at 37xc2x0 C. for 2.5 hours. The plates are then spun briefly by centrifugation at 800xc3x97g for 5 minutes, medium is aspirated from wells, and 50 xcexcl ethanol/well is added to dissolve the formazan formed during the incubation period with MTT. After a 15 minute shaking, the optical density is determined for each well in an automatic plate reader at 570 and 660 nm. Percent inhibition of cell growth is calculated by comparing optical densities of cells treated with test compounds to those of cells treated only with 0.1% ethanol. IC50 values are determined based on the Reed and Muench formula (Reed, L. J., and H. Muench, A simple method of estimating fifty percent endpoint. Am. J. Hyg. 27:493-497 (1938)) and the results are presented below in Table II.
The useful activity of compounds of formula I as agents for the treatment of hyperproliferative skin disease can be demonstrated by the following.
Inhibition of Keratinocytes Proliferation
HaCaT cell linexe2x80x94The immortalized human cell line HaCaT was used. 3H-thymidine incorporation was measured in exponentially growing cultures after 6 days of culture in presence of the test compound.
Cell culturexe2x80x94HaCaT cells were cultured in Dulbecco""s Modified Eagle Medium (DMEM) and Nutrient Mixture Ham""s F12 (F12), 3:1 (v/v, ICN) containing 4.5 g/l glucose and supplemented with 10% fetal calf serum (Gibco, FCS), L-glutamine (Gibco, 2 mM), penicillin (Gibco, 50 UI/ml), streptomycin (Gibco, 50/xcexcg/ml), EGF (10 ng/ml), hydrocortisone (400 ng/ml), cholera toxin (8.5 ng/ml) and insulin (5 ng/ml). The cells were maintained in a humidified atmosphere containing 5% CO2 and 95% air and passaged every 3-4 days.
Inhibition of 3H-thymidine uptake xe2x80x94HaCaT cells (250 cells in complete culture medium) were seeded into 96-well culture dishes and incubated at 37xc2x0 C. with 5% CO2 and 95% air for 6 days. Inhibitors, dissolved at 10xc3x97concentration in 1% ethanol, were added immediately at the beginning of the assay. 3H-thymidine (5 Ci/mmol, Amersham) was added at a concentration of 1 xcexcCi/well and cells were pulse-labeled for the last 6 hours of the growth period. Cells were then trypsinized for 10 minutes at 37xc2x0 C. under a vigorous agitation and harvested on to a 96-well GF/C filter plate (Uni Filter, Packard) using a Micro Mate 196 cell harvester (Packard). After drying at 40xc2x0 C. under vacuum for 20-30 minutes, 20 xcexcl of Micro Scint 0 scintillator (Packard) were added and the radioactivity bound to the filters was counted on a TOP COUNT (Packard). The results are set forth in Table III.
The useful activity of compounds of formula I as agents for the treatment of sebaceous gland diseases can be demonstrated by the following.
Inhibition of Human Sebocyte Proliferation In Vitro
Sebaceous cells were isolated from adult human sebaceous glands by a combination of enzymatic and mechanical methods (Doran et al., Characterization Of Human Sebaceous Cells In Vitro, J. Invest. Dermatol. 96:341-8 (1991)). The cells were cultured in Iscove""s medium containing 10% fetal calf serum and 4 xcexcg/ml dexamethasone on a layer of growth-arrested 3T3 mouse fibroblasts. Cells were plated in medium without the test compound and then given test compound in fresh medium 24-48 hours after the initial plating. The cultures were given fresh medium, containing the test compound, every 48 hours. On the day of harvesting, the cultures were rinsed with 0.03% EDTA (ethylenediamine tetroacetic acid) in PBS (phosphate-buffered saline), to remove only the 3T3 fibroblasts, followed by incubation in 0.05% trypsin/0.03% EDTA. The cells were suspended, mixed vigorously to prepare a single cell suspension and counted in a hemocytometer. Stock solutions of compounds were made up as 10xe2x88x922 M solutions in degassed 100% ethanol and stored at xe2x88x9220xc2x0 C. in the dark. During experimental use, the solutions, which have been aliquoted, were brought to room temperature and used by diluting directly into complete medium to the appropriate concentration.
The compounds were tested for the inhibition of proliferation of sebaceous cell growth in vitro at 10xe2x88x926, 10xe2x88x927 and 10xe2x88x928 M. The results are summarized in Table IV as the amount of compound necessary to inhibit the proliferation of sebaceous cells by 50% (ED50) in nM as compared to a vehicle-treated culture.
Calcium Tolerance Test in Mice
Profound changes in calcium homeostasis strongly affect the weight development of mice.
Mice (25-30 g body weight) received daily subcutaneous administrations of the compound for 4 consecutive days. Body weight was registered just before and at the end of a 5 day treatment period. The xe2x80x9chighest tolerated dosexe2x80x9d (HTD) is the dose which results in zero weight gain during this treatment period. The results are set forth in Table V.
The following Examples are provided to further describe the invention and are not intended to limit it in any way.